Cell Direct RT qPCR Kit—Taqman Direct Cell Lysis Cell Ready Gawo limodzi qRT-PCR Kits Probe
Kufotokozera
Izi zimagwiritsa ntchito njira yapadera ya lysis buffer kuti itulutse mwamsanga RNA kuchokera ku maselo opangidwa ndi chikhalidwe cha RT-qPCR, kuthetsa nthawi yowonongeka ndi yolemetsa ya RNA, ndi mphindi 7 zokha kuti mupeze template yofunikira ya RNA, ndi 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman yoperekedwa ndi zida za nthawi yeniyeni ya PCR.
5 × Direct RT Mix ndi 2 × Direct qPCR Mix-Taqman ali ndi kulekerera kwamphamvu kwa inhibitor ndipo amatha kuchita bwino kusinthika ndi kukulitsa kwachindunji pogwiritsa ntchito lysate yachitsanzo kuti chiyezedwe ngati template.The reagent ili ndi Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl.2, Reaction Buffer, PCR Optimizer ndi Stabilizer, yomwe ingagwiritsidwe ntchito ndi lysis buffer kuti izindikire mwamsanga komanso mosavuta zitsanzo, ndipo imakhala ndi makhalidwe apamwamba, kukhazikika komanso kukhazikika.
Zofotokozera
200×20μl Rxn, 1000×20μl rxns
Zida zamagulu
| QuickEasyTMCell Direct RT-qPCR Kit-Taqman | ||||
| Zida zamagulu 20μl qPCR Reaction System | DRT-01021 | DRT-01022 | Zindikirani | |
| 200 T | 1000 T | |||
|
Gawo I | Mtengo CL | 4 ml pa | 20 ml | Cell Lysis |
| Foregene Protease Plus II | 80 ml | 400 μl | ||
| Mtengo wapatali wa magawo ST | 400 μl | 1 ml × 2 | ||
|
Gawo II | DNA Eraser | 80 ml | 400 μl | |
| 5×Direct RT Mix * | 160 μl | 800 ml | RT | |
| 2 × Direct qPCR Mix-Taqman * | 1 ml × 2 | 1.7ml × 6 | qPCR | |
| 20 × ROX Reference Dye | 40 ml | 200 μl | ||
| RNase-Free ddH2O | 1.7 ml | 10 ml | ||
| Buku Lolangiza | 1 chidutswa | 1 chidutswa | ||
*:Cell Lysis, 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman zitha kugulidwa padera
Mbali & ubwino
■Zosavuta komanso zothandiza : ndiukadaulo wa Cell Direct RT, zitsanzo za RNA zitha kupezeka m'mphindi 7 zokha.
■ Zofuna zachitsanzo ndizochepa, mpaka ma cell 10 akhoza kuyesedwa.
■ Kutulutsa kwakukulu: kumatha kuzindikira mwachangu RNA m'maselo opangidwa mu 384, 96, 24, 12, 6-chitsime mbale.
■ DNA chofufutira akhoza kuchotsa mwamsanga matupi athu omasulidwa, kwambiri kuchepetsa zotsatira zoyeserera.
■ Makina okhathamiritsa a RT ndi qPCR amapangitsa kuti masitepe awiri a RT-PCR alembedwe bwino kwambiri komanso PCR yachindunji, komanso yosagwirizana ndi RT-qPCR reaction inhibitors.
Kit application
Kuchuluka kwa ntchito: maselo otukuka.
- RNA yotulutsidwa ndi zitsanzo za lysis: imagwira ntchito pa template ya RT-qPCR ya zida izi.
- Zidazi zitha kugwiritsidwa ntchito pazifukwa izi: kusanthula kwa jini, kutsimikizira kwa siRNA-mediated gene silence effect, kuyang'anira mankhwala, etc.
Kusungirako ndi moyo wa alumali
Gawo I la zida izi liyenera kusungidwa pa 4℃;Gawo II liyenera kusungidwa pa -20 ℃.
Foregene Protease Plus II iyenera kusungidwa pa 4℃, osazizira pa -20 ℃.
Reagent 2×Direct qPCR Mix-Taqman iyenera kusungidwa pa -20℃mumdima;ngati imagwiritsidwa ntchito pafupipafupi, imathanso kusungidwa pa 4℃ posungira kwakanthawi kochepa (gwiritsani ntchito mkati mwa masiku 10).
Real Time PCR primer design mfundo
Forward Primer ndi Reverse Primer
Kwa Real Time PCR, mapangidwe oyambira ndi ofunika kwambiri.Zoyambira zimayenderana ndi kutsimikizika komanso kuthekera kwa kukulitsa kwa PCR, ndipo zitha kupangidwa motengera mfundo izi:
- Kutalika koyambirira: 18-30bp.
- Zolemba za GC: 40-60%.
- Mtengo wa Tm: Mapulogalamu opangira ma primer, monga Primer 5, amatha kupereka mtengo wa Tm woyambira.Makhalidwe a Tm oyambira kumtunda ndi kumtunda ayenera kukhala pafupi momwe angathere.Njira yowerengera ya Tm ingagwiritsidwenso ntchito: Tm = 4 °C (G + C) + 2 °C (A + T).Pochita PCR, kutentha pansi pa mtengo woyambira wa Tm wa 5 ° C nthawi zambiri kumasankhidwa ngati kutentha kwapang'onopang'ono (kuwonjezeka kofananirako kwa kutentha kwapang'onopang'ono kumatha kukulitsa momwe PCR imachitira).
- Primers ndi zinthu za PCR:
- Design primer PCR amplification product kutalika makamaka 100-150bp.
- Zoyambira zojambula m'dera lachiwiri la template ziyenera kupewedwa momwe zingathere.
- Pewani kupanga zoyambira ziwiri kapena kupitilira apo pakati pa 3′ malekezero oyambira kumtunda ndi kumunsi kwa mtsinje.
- Primer 3′ terminal base sangakhalepo ndi 3 zowonjezera zotsatizana za G kapena C.
- Zoyambira zokha sizingakhale ndi zida zowonjezera, apo ayi mawonekedwe atsitsi adzapangidwa, zomwe zimakhudza kukulitsa kwa PCR.
- ATCG iyenera kugawidwa mofanana momwe mungathere poyambira, ndipo 3′ terminal base iyenera kupewedwa ngati T.
Zowonjezera1:Cndi DirectRT-qPCR Kit chigawot paketi yowonjezera
1.Cell Lysis Solution
| Cell Lysis Solution | |||
| Zida zamagulu (24-chitsime lysis dongosolo / bwino) | Chithunzi cha DRT-01011-A1 | Chithunzi cha DRT-01011-A2 | |
| 100 T | 500 T | ||
| GawoIne | Mtengo CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Mtengo wapatali wa magawo ST | 1 ml × 2 | 10 ml | |
| GawoII | DNA Eraser | 400 μl | 1 ml × 2 |
| RT Mix | |
| Zida zamagulu (20 μl reaction system) | Chithunzi cha DRT-01011-B1 |
| 200 T | |
| 5 × Direct RT Mix | 800 ml |
| RNase-Free ddH2O | 1.7ml × 2 |
| qPCR Mix | ||
| Zida zamagulu (20 μl reaction system) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2 × Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7ml × 6 |
| 20 × ROX Reference Dye | 40 ml | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Mabuku a Malangizo:
